Project Summary Hematopoietic stem and progenitor cells (HSPCs) are self-renewing, transplantable cells that support lifelong blood production. HSPC function and transplantation are tied to signal transduction through G-protein coupled receptors (GPCRs), particularly that of CXCR4 which regulates homing and niche retention. Our understanding of basic GPCR regulatory mechanisms in these cells are lacking. This is highlighted by our laboratories recent finding that two GPCR-trafficking proteins, GPRASP1 and GPRASP2, act as negative regulators of HSPC transplantation. Our preliminary data further indicate that these proteins limit HSPC quiescence and resistance to apoptosis in a CXCR4-dependent manner. GPRASP1 and -2 have been described in the nervous system to promote the lysosomal degradation and functional downregulation of opioid, dopamine, and other select receptors. The receptors regulated by GPRASP1 and -2 in HSCs are currently unknown. As a postdoctoral fellow in the McKinney-Freeman laboratory, I will investigate the molecular functions of GPRASP1 and -2 in HSPCs. In Aim 1, I will use biochemical methods to definethe impact of GPRASP1 and -2 on CXCR4 signaling. The impact of GPRASP1 and -2 on CXCR4 endocytosis, degradation, and downstream signaling outcomes after agonist stimulation will be analyzed in mouse HSPCs. In Aim 2, I will probe the role of GPRASP1 and -2 in transplantation of human HSPCs isolated from cord blood. Human HSPCs that have undergone gene editing by CRISPR/Cas9 nucleofection to knockout GPRASP1 or -2 and will be used in competitive xenotransplantation assays. By following the relative engraftment and blood production of wild type and knockout human HSPCs after xenotransplantation the impact of GPRASP proteins on this procedure will be determined. In Aim 3, I will use an unbiased proteomics approach to investigate the receptor targets of GPRASP1 and -2, as well as the downstream trafficking pathways associated with these proteins. Biotin proximity labeling will be performed using GPRASP-APEX fusion proteins in K562 chronic myelogenous leukemia cells both before and at multiple timepoints following agonist stimulation. Biotinylated samples will undergo Tandem Mass Tagging and multiplex mass spectrometry to determine the relative abundance of proteins across timepoints. This will allow for bioinformatic analysis of the receptor targets and downstream trafficking pathways associated with GPRASP1 and -2. All protein associations of interest will be validated by immunofluorescence in mouse HSPCs. These aims draw on my prior training as a biochemist but provide abundant training opportunities in the field of HSPC biology. The McKinney-Freeman laboratory and St. Jude are ideal environments in which to receive training in HSPC biology. Institutional resources provided, including state-of-the-art flow cytometry and proteomics cores, will greatly contribute to the success of this project. In addition, I will take advantage of numerous career development resources during my fellowship. These resources include scientific writing workshops, presentation and networking opportunities, and teaching and mentoring experiences.